This project is focused on nuclear receptor regulation of metabolic and inflammatory gene expression and the impact of nuclear receptor signaling on cardiovascular disease. Work from this project over the past ten years has established that LXRs function as transcriptional regulators of cholesterol metabolism and modulators of immunity and inflammation. Studies in the current grant period have revealed novel mechanisms whereby cholesterol metabolism may impact immune functions of macrophages. We identified new LXR target genes (arginase II and Mertk) that regulate macrophage inflammatory and phagocytic capacity. In addition, we provided in vivo evidence that the LXR signaling pathway is an important determinant of atherosclerosis susceptibility and immune tolerance. Finally, we identified a novel mechanism whereby LXR activation regulates cholesterol uptake through the LDL receptor pathway. We identified an E3 ubiquitin ligase termed Idol that targets the LDLR for degradation in response to LXR activation. In the next funding period we will extend this work by investigating the mechanism and physiologic function of the LXR-Idol-LDLR axis in cellular cholesterol uptake. We will also utilize mouse models to determine the function of Idol in systemic lipid metabolism and its impact on atherosclerosis. These studies are expected to provide insight into novel mechanisms by which LXR and Idol regulate cholesterol homeostasis and atherosclerosis and may identify additional targets for intervention in cardiovascular disease. Specific Aim 1 is to use in vitro models to define the function of the LXR-Idol-LDLR axis in cellular cholesterol homeostasis. We will utilize gain and loss of function systems to determine the impact of Idol expression on cholesterol uptake, metabolic gene expression, and potential crosstalk with the SREBP signaling pathway. We will also use site-directed mutagenesis and functional assays to define the regions of Idol and LDLR important for Idol-LDLR recognition and ubiquitination. Specific Aim 2 is to use in vivo models to define the function of the LXR-Idol-LDLR axis in systemic lipid metabolism. We have generated transgenic animals that express Idol from the liver-specific albumin promoter. We have also generated mice carrying a global deletion in the Idol gene. We will use these models to examine the ability of Idol to affect plasma cholesterol levels, as well as its influence on hepatic and peripheral cholesterol metabolism. Specific Aim 3 is to determine the impact of Idol on macrophage lipid homeostasis and the development of atherosclerosis. We will use gain- and loss-of-function approaches to test the hypothesis that the LXR-Idol pathway is a physiologic limiter of macrophage cholesterol accumulation. To test the relevance of the macrophage Idol pathway for atherogenesis, we will analyze lesion formation in LDLR-/- mice transplanted with WT or Idol-/- bone marrow. We will also generate and analyze lesion formation in Idol-/- human apoBTg mice. PUBLIC HEALTH RELEVANCE: A detailed understanding of nuclear receptor function in macrophages and other cells is expected to provide insight into basic molecular mechanisms controlling lipid metabolism and inflammation as well as the pathobiology of atherosclerosis. Our identification in the current funding period of Idol, a specific mediator of LDLR degradation, provides interesting new insight into the LDLR pathway, known to be a major contributor to human cardiovascular disease risk. Ultimately, the studies proposed in this application may form the basis for the development of new diagnostic and therapeutic approaches to cardiovascular disease.